The long range goal of the proposed research is to understand the contributions of ankyrin and spectrin (the membrane skeleton) to plasma membrane assembly and function in higher organisms. Human genetic studies have made an important contribution to our understanding of the membrane skeleton as a link between transmembrane proteins and actin. But these studies are constrained to specialized protein isoforms that have a limited tissue distribution. Genetic studies of the more widely expressed membrane skeleton isoforms have not been possible in mammals. Drosophila will be used as a model system where powerful molecular and genetic tools are available for the study of protein function in a complex developing organism. We have identified and partially characterized a Drosophila ankyrin homolog that is conserved in size, sequence, and spectrin interaction, relative to its human counterpart. Mutations in Drosophila ankyrin will be identified and characterized. The phenotype of ankyrin null mutants will reveal the cellular consequences of ankyrin deficiency in a range of different cell types. Molecular analysis of ankyrin mutations will provide new information on functional sites within the ankyrin molecule. A transgene rescue approach with ankyrin cDNA constructs will be established to verify that defects in ankyrin explain the observed phenotypes of mutants. Rescue experiments with modified ankyrin transgenes will be used to test the functional contribution of selected sites in the ankyrin molecule. A unique advantage of Drosophila as a model system is the availability of mutants that are defective in several of the proteins that interact with ankyrin, such as the sodium pump sodium channels and cell adhesion molecules. Simple bioassays that reflect the functional levels of these proteins will be used to study the consequences of membrane skeleton defects. The levels of these interacting proteins will also be manipulated in order to study their effects on membrane skeleton assembly. These experiments will provide the first direct tests of the predicted functions of ankyrin that have emerged from descriptive studies in mammalian systems. Studies of erythrocyte ankyrin have established the importance of this membrane skeleton protein in human disease. Current questions concern the unknown role of ankyrins in non-erythroid cells. The results of the proposed experiments will provide new insights into the role of non- erythroid ankyrins and will significantly extend the current understanding of ankyrin function in humans.